HK1066861B - Method of measuring whole blood - Google Patents
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- HK1066861B HK1066861B HK04109808.9A HK04109808A HK1066861B HK 1066861 B HK1066861 B HK 1066861B HK 04109808 A HK04109808 A HK 04109808A HK 1066861 B HK1066861 B HK 1066861B
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Description
Technical Field
The present invention relates to a method for analyzing a specific component contained in whole blood using whole blood as a sample.
Background
Components in blood such as antigens, antibodies, proteins, endocrine substances, and the like are clinically very important. Generally, in many cases, plasma or serum is used as a blood sample, and in such cases, in order to avoid hemolysis, whole blood is generally separated into serum or plasma as quickly as possible. This is because, for example, in the field of immunoassay, if a blood cell component is present, or hemolysis is caused in a sample, a phenomenon may occur which causes disturbance such as affecting hemolysis on the visual system, suppressing immune reaction by an internal component of a blood cell, and aggregation or adhesion of a plasma membrane component of a blood cell on a solid carrier serving as a solid phase. Therefore, in a common clinical test, it has been a general practice to first centrifuge whole blood to remove blood cells and to use the resulting plasma or serum as a sample for testing.
However, in order to remove blood cells, a special instrument such as a centrifuge is required, and the operation is also labor-consuming. Therefore, the use of whole blood as a test sample is desirable for technicians who do not have such equipment in actual practice and who perform urgent tests with little temporary gaps.
In order to meet the above need, various methods of testing whole blood without separating serum or plasma have been proposed. As an immunoassay, a method using latex agglutination as a homogenization test (method not requiring B/F separation) has been reported as a test method which is a deliberate and forced cause of hemolysis of blood cells (Japanese patent Laid-open (kokai), 10-48214). Secondly, as a test method not causing hemolysis of blood cells, a homogeneous test method using latex dispersed light (clinical chemistry, vol. 43, 1764-1770(1997)), a heterogeneous test (heterogeneous assay) method using a plastic tubule as a solid phase (a method requiring B/F separation) (Japanese patent laid-open No. 6-265554), and a method using polypropylene beads or magnetic particles as a solid phase (Japanese International patent laid-open (kohyo), 2000-508075, WO96/04558) have been reported.
However, it cannot be said that with these methods, a convenient and highly sensitive test method using whole blood as a sample has been established. First, some immunoassay methods using a homogeneous test have been reported as convenient methods, but analytes are often trace substances and the like contained in blood in clinical tests and the like, so it is often highly desirable to test whole blood using a heterogeneous test method which is a theoretically highly sensitive test method. Secondly, with the background that solid carriers such as magnetic particles are widely used for heterogeneous tests as a solid phase due to the simplicity of B/F separation, microparticles such as magnetic particles seem to be affected particularly by blood cells, although solid carriers having such a size that the solid carriers will not aggregate do not cause problems as in the case of beads and plastic plates having diameters of the order of micrometers. For example, when hemolysis occurs, inhibitory substances such as hemoglobin and nuclear derivatives flowing out of the interior of blood cells into the reaction system may cause non-specific agglutination of solid carriers or reduce immune reactions, thereby seriously affecting the test. In addition, even when fresh unhemolyzed whole blood is used as a sample, if blood cells are present, the solid support seems to become liable to adhere on the inner wall of the reaction vessel or on the tip of the dropping tube because of substances on the cell membrane surface of the blood, etc., and therefore, may cause damage such as inaccurate testing.
In addition, automated testing instruments and cartridges (cartridges) are often utilized to enable the rapid and convenient performance of whole blood tests as described above. However, the same problem occurs in each step in such automatic testing. That is, the blood cell component in the whole blood and the solid carrier used in the test are precipitated with time, and therefore it is necessary to include a step of efficiently stirring the sample containing the whole blood before the test to maintain the blood cell component uniform, or a step of efficiently stirring the sample, the solid carrier, the reagent, and the like in the reaction step or the test step. In such a stirring step, a strong force is applied to the blood cells, and the blood cells are destroyed, resulting in extreme susceptibility to hemolysis. In addition, since the aspiration and the discharge of the sample are performed at each step so as to successfully transfer the sample to the target reaction vessel according to the step, a strong force is applied to the blood cells, so that hemolysis is easily generated. In addition, nonspecific adhesion and aggregation also easily occur. Therefore, test errors are often caused.
In recent years, such instruments and films in automatic testing as described above are also widely used in the field of point of care testing (POCT), and they have attracted attention as a burst test method or a test method easily performed by technicians and nurses. Therefore, there is a need to develop a test method that can provide accurate test results even with such an instrument and whole blood.
Disclosure of Invention
It is an object of the present invention to provide a rapid and convenient method for testing an analyte in whole blood, which is highly sensitive, using whole blood as a sample.
The inventors of the present invention have made various studies in order to achieve the aforementioned object. As a result, they found that, in a method of testing an analyte contained in a sample containing whole blood, if a reaction is performed in the presence of a detergent and in a state in which blood cells are not destroyed, the test can be performed in a short period of time, the sensitivity is high, and separation of serum/plasma by centrifugation or the like is not performed.
Accordingly, the present invention provides a method for testing an analyte, which comprises a reaction step of forming a reaction system comprising a sample containing whole blood, a first substance carried by magnetic particles and specifically binding to the analyte contained in the sample, and a second substance labeled with an enzyme or a luminescent substance and specifically binding to the analyte, and reacting the analyte with the first and second substances to form a reaction product, a separation step of separating the reaction product formed, and a measurement step of measuring the separated reaction product, wherein:
(1) the reaction step is carried out in a state where the blood cells are not destroyed; and
(2) at least the reaction step is carried out in the presence of a detergent in an amount sufficient not to cause hemolysis, not to inhibit the reaction of the analyte with the first and second substances that specifically bind to the analyte, and to prevent the influence of components present in the reaction system on the reaction system.
In addition, another embodiment of the invention is a method of determining an analyte in whole blood, comprising:
(1) a dilution step of diluting whole blood by mixing the whole blood with a whole blood treatment solution;
(2) a first reaction step of adding a first substance carried by magnetic particles and specifically binding to an analyte to diluted whole blood to react them to form a first reaction product in a reaction system;
(3) a first separation step of separating a first reaction product formed in the first reaction step from the reaction system;
(4) a second reaction step of adding a second substance labeled with an enzyme or a luminescent substance and specifically binding to the analyte to the separated first reaction product and reacting them to form a second reaction product in the reaction system;
(5) a second separation step of separating a second reaction product formed in the second reaction step from the reaction system; and
(6) a measuring step of measuring the separated second reaction product, wherein
The whole blood treatment solution contains a sufficient amount of a detergent that does not cause hemolysis when the solution is mixed with whole blood, does not inhibit the reaction of the analyte with the first and second substances, and can prevent the influence of the components present in the reaction system in each step on the reaction system.
In addition, in another aspect of the present invention, a kit for use in the assay method of the present invention is provided. An example of the kit is a kit for testing an analyte in whole blood, which comprises a first substance carried by magnetic particles and specifically binding to the analyte, a second substance labeled with an enzyme or a luminescent substance and specifically binding to the analyte, and a detergent which does not cause hemolysis when mixed with the whole blood and does not inhibit the reaction of the analyte with the first substance and the second substance.
Therefore, the present invention will be explained in detail below.
The test method of the present invention is a method of testing a sample containing whole blood for an analyte.
The term "whole blood-containing sample" refers to whole blood collected from a patient, whole blood mixed with some processing solution (also referred to as "whole blood processing solution" as previously described), and the like. The term "whole blood" refers to whole blood collected from a patient under the assumption that it contains an analyte or may contain an analyte, and preferably fresh blood used within 3 days after collection, more preferably within 24 hours after collection, or more preferably immediately after collection, or within 12 hours after collection. Blood can be collected by a blood collection tube or the like by a known method, and treated with an anti-coagulating agent such as EDTA or heparin or the like. The blood is preferably refrigerated, more preferably stored at 4 to 0 ℃.
The analyte is not particularly limited as long as it is contained in whole blood and is a substance that specifically binds to the substance to form a reaction product. Examples of the combination of the analyte and the specifically binding substance include antigens and antibodies, antibodies and antigens, proteins and ligands, sugar chains and lectins, and the like. Particularly preferred are antigens and antibodies or antibodies and antigens. Therefore, in the present invention, the term "specifically binds" refers to the formation of a reaction product by a biochemical specific bond. Specific examples of the analyte include hepatitis B virus surface antigen (HBsAg), Hepatitis C Virus (HCV) antibodies and antigens, Human Immunodeficiency Virus (HIV) antibodies, human T-cell leukemia virus-1 (HTLV-1) antibodies, Treponema Pallidum (TP) antibodies, and the like. In addition, various cardiac markers (creatine kinase (CKMB), myoglobin, troponin), various hormones, serum proteins, and the like are also possible.
In addition, the test method of the present invention is a heterogeneous test method using a first substance that specifically binds to an analyte and a second substance that specifically binds to an analyte, which are carried on a solid support. Such a method may be any method as long as it includes a reaction step of reacting the aforementioned analyte with the first and second substances in a sample containing whole blood and a measurement step of measuring a reaction product formed.
Specifically, a reaction system is formed which includes the aforementioned sample, a first substance carried by the solid support and specifically binding to the analyte, and a second substance specifically binding to the analyte, and the analyte reacts with the first and second substances. Although the first and second species may react with the analyte simultaneously or sequentially, preferably sequentially. In the former embodiment, for example, the first substance and the second substance are added to the sample. In the latter embodiment, the method comprises two reaction steps, for example, a first reaction step in which a first substance is added to the sample and allowed to react to form a first reaction product, and a second reaction step in which a second substance is added to the first reaction product and allowed to react to form a second reaction product. Therefore, in the present invention, the expression "forming a reaction system including a sample, a first substance, and a second substance" includes an example in which three components are reacted simultaneously (i.e., including one reaction step) and an example in which three components are reacted sequentially (i.e., including two reaction steps).
After the first reaction step in which the analyte reacts with the first substance to form a first reaction product, B/F separation is preferably performed (first separation step). In addition, a second reaction step of reacting the second substance with the first reaction product is performed after the B/F separation, and then preferably a second B/F separation (second separation step) is performed. By such an operation, the measurement can be performed with higher sensitivity. The conditions in each of these steps may be appropriately selected depending on the combination of the analyte and the substance specifically binding thereto.
Specifically, for example, when an antibody and an antigen react and the amount of the reaction product is measured, the measurement can be performed as follows. That is, an antigen or an antibody contained in whole blood, a solid carrier carrying the antibody or the antigen (first substance) specifically binding thereto, and another labeled antibody or antigen (second substance) are mixed to form an immune complex. Then, unreacted antibody and antigen were removed by washing (B/F separation), and the amount of the labeling substance bound to the solid support was measured. More specifically, for example, a sample containing whole blood and magnetic particles (solid carrier) carrying a first substance are placed in a reaction vessel and stirred, and then, an antigen-antibody reaction is performed at a predetermined temperature for a predetermined time. After the reaction, unreacted materials were removed from the reaction vessel by B/F separation using magnetic force. Subsequently, the labeled second substance is placed in the reaction vessel, reacted at a predetermined temperature for a predetermined time, and B/F separation is performed again using a magnetic force to remove the unreacted substance. Finally, the amount of the analyte can be determined by measuring the amount of the labeling substance contained in the reaction product produced.
The solid support is not particularly limited as long as it is substantially insoluble in various solutions used in the assay. However, it is preferable to use magnetic particles, polymers such as polystyrene or latex thereof, gelatin, liposomes, and the like. Among them, magnetic particles are particularly preferable from the viewpoint of achieving rapid and simple B/F separation. Specific examples thereof include magnetic particles composed of microparticles of a metal such as ferroferric oxide (Fe)3O4) Iron oxide (Fe)2O3) Various kinds of ferrite, iron, magnesium, nickel, cobalt and chromium, and alloys of cobalt, nickel, magnesium, and the like. In addition, it is also preferable that these magnetic particles are contained in a latex of a polymer such as polystyrene, gelatin, liposome, or the like, or are immobilized on the surface of these substances.
The particle size of these solid carriers is not particularly limited as long as the B/F separation can be accurately performed. However, too small a particle size results in a low separation efficiency, so aggregation easily occurs. On the other hand, an excessively large particle size easily causes precipitation. Therefore, the lower limit of the particle size is 0.05. mu.m, preferably 0.1. mu.m, and the upper limit is suitably 10 μm, preferably 4 μm, more preferably 2 μm. The particle size range is determined by a combination of these upper and lower limits. The specific particle size range of the carrier is usually 0.05 to 10 μm, preferably 0.05 to 4 μm, more preferably 0.1 to 2 μm.
The first substance specifically binding to the analyte can be carried by such a solid support using known methods. Specifically, for example, a chemical bonding method, a physical adhesion method, or the like can be used.
In the test method, B/F separation using the solid support prepared as described above may be performed by a filtration method, an antibody capture technique, a precipitation method, or the like. Particularly, when magnetic particles are used, a magnetic field may be generated using a permanent magnet, an electromagnet, or the like to rapidly and conveniently perform B/F separation using a magnetic force.
The test method of the present invention is characterized in that (1) the aforementioned reaction step is carried out in a state where blood cells are not destroyed, and (2) at least the reaction step is carried out in the presence of a detergent which is large enough not to cause hemolysis and not to inhibit the reaction of an analyte with first and second substances which specifically bind to the analyte, and which can prevent the influence of components present in the reaction system on the reaction system.
The expression "state in which blood cells are not destroyed" is not limited as long as the reaction step can be performed without destroying blood cells in whole blood. This state means a state in which blood cells are not destroyed, or a state in which a small number of blood cells are destroyed but not enough to affect the test. As a method for achieving a state in which blood cells are not destroyed, there are a method of adding a detergent which does not cause hemolysis in a reaction system, a method of adjusting the isotonic pressure of a reaction system with an isotonic solution such as physiological salt, a method of adding magnesium ions or the like to a reaction system to prevent the rupture of cell nuclei or the like. In addition, these methods may be used in combination.
The detergent used in the present invention is not particularly limited as long as it is a concentration and a type that do not cause hemolysis, do not inhibit specific binding of the analyte and the first and second substances to the analyte, and can prevent influence of components present in the reaction system on the reaction system. The expression "not causing hemolysis" as used herein means that the detergent does not cause hemolysis or that the hemolysis is so weak that the test is not affected when mixed with a sample contained in whole blood. The expression "does not inhibit the reaction of the analyte with the first and second substances which specifically bind to the analyte" means that the detergent does not inhibit the formation of a reaction product by biochemical specific binding of these substances, or inhibits so weakly that the assay is not affected. The expression "preventing the influence of the components present in the reaction system on the reaction system" means that the detergent inhibits non-specific aggregation, adhesion, binding other than target-specific binding, etc. on the inner wall of the reaction vessel or pipette tip, etc. caused by blood cells, other components or the like present in the reaction system, thereby preventing their influence during the reaction step.
As described above, by adding a detergent to the reaction system, hemolysis can be prevented, non-specific adhesion of a solid carrier such as magnetic particles to the inner wall of the reaction vessel or pipette tip can be prevented, and the influence of blood cell components and blood cells during the measurement can be avoided, whereby accurate measurement can be carried out.
In the present invention, a polyoxyethylene sorbitan ester type or sulfobetaine type detergent is particularly preferably used.
The polyoxyethylene sorbitan ester-based detergent includes polyoxyethylene sorbitan monolaurate (Tween 20), polyoxyethylene sorbitan monooleate (Tween 80), and the like. Among these, polyoxyethylene sorbitan monooleate (Tween 80) has a weak hemolytic action and is preferably used.
Examples of sulfobetaine-type detergents include: dimethylethylammonium propanesulfonate, 3- (1-pyrido) -1-propanesulfonate, dimethylbenzylammonium propanesulfonate, N-octyl-N, N-dimethyl-3-amino-1-propanesulfonate, N-decyl-N, N-dimethyl-3-amino-1-propanesulfonate, N-dodecyl-N, N-dimethyl-3-amino-1-propanesulfonate, N-tetradecyl-N, N-dimethyl-3-amino-1-propanesulfonate, N-hexadecyl-N, N-dimethyl-3-amino-1-propanesulfonate, and the like. Of these, dimethylethylammonium propanesulfonate, 3- (1-pyrido) -1-propanesulfonate, dimethylbenzylammonium propanesulfonate and N-octyl-N, N-dimethyl-3-amino-1-propanesulfonate have a weak hemolytic action and are preferably used.
Before the reaction step of forming a reaction system including the sample, the first and second substances that specifically bind to the analyte contained in the sample, and reacting the analyte with the first and second substances, as a pretreatment, a detergent may be added to the whole blood treatment solution to be mixed with the whole blood. However, since the aforementioned detergent does not substantially inhibit the reaction of the analyte with the specifically bound first and second substances, for example, when a solution of an antibody immobilized on a solid phase is used as the first substance carried by the solid support, the detergent may be added to the solution in advance so as to allow the solution to directly react with the sample containing whole blood. In addition, it is sufficient to add a detergent to at least the first reaction step, in which a plurality of blood cells are contained in the reaction mixture. However, since they also have the effect of inhibiting non-specific adhesion or aggregation of the solid carrier, it is preferable to add a detergent also in the second reaction step. They may be added at all steps including the testing step.
Such detergents may be added at any concentration as long as they are added at such a concentration that the aforementioned effects can be exerted. Specifically, they are added in the reaction step in a final concentration range of, for example, 0.1 to 10%, preferably 0.5 to 5%, more preferably 0.5 to 2%. One such type of detergent may be added alone, or a mixture of two or more types may be used. When mixtures of two or more types are used, they may also be present in any combination within the range in which the aforementioned effects are exerted. In addition, when a detergent is used in the whole blood solution, the whole blood treatment solution may be prepared so that the concentration of the detergent ranges from 0.1 to 50%, preferably from 0.5 to 30%. The ratio of the whole blood treatment solution containing the detergent prepared as described above may be such that the concentration of the detergent in the whole blood-containing sample after mixing is within the aforementioned concentration range. In addition, the ratio of mixing is preferably determined in consideration of the amount of the analyte contained in the sample. When a trace amount of substance contained in a sample of a small amount is to be measured, the proportion of the whole blood treatment solution is preferably measured to be small. Specifically, for example, the mixing ratio of the whole blood and the whole blood treatment solution may be in the range of 99: 1 to 5: 95.
The whole blood treatment solution used in the present invention may be arbitrarily selected and used as long as the amount or characteristic of the solution is such that the blood cell component in the whole blood is not hemolyzed. Or various components are not denatured. Specific examples include solutions for adjusting physiological pH, isotonic pressure, salt concentration and the like, such as phosphate buffer solution (phosphate buffered saline; PBS), physiological saline solution. In addition, any solution other than the solution prepared as described above may also be mixed as long as its amount is such that blood cell components and other components are not affected. However, if the analyte is only a substance having a small content in whole blood, it is preferable to perform the test with whole blood itself or by mixing whole blood with a whole blood treatment solution in a low mixing ratio.
The second substance is preferably labelled. Examples of the labeling substance include an enzyme, a luminescent substance, a fluorescent substance, a radioisotope, a coloring substance, various colored particles, and the like. Among them, enzymes are preferably used. Examples of enzymes commonly used in chemiluminescent enzyme immunoassay (CLEIA) include alkaline phosphatase, peroxidase, galactosidase, glucose oxidase (glucooxidase), and the like. As substrates for these enzymes, substrates corresponding to these enzymes are selectable. For example, adamantyl methoxyphenyl phosphoryl dioxetane (AMPPD) may be used for alkaline phosphatase, luminol/peroxide may be used for peroxidase, and adamantyl methoxyphenyl- β -D-galactosyl dioxetane (AMPGD) may be used for galactosidase.
As a method for measuring the reaction product, any conventional method can be used. For example, when a second substance labeled as described above is used, the determination can be conveniently carried out by determining the amount of the labeled product in the reaction product. For example, when a chemiluminescent enzyme immunoassay (CLEIA) method is used, the luminescence intensity of a labeling substance in the reaction product can be measured using a photomultiplier tube (PMT) or the like.
That is, in the present invention, the expression "measuring the reaction product" means not only directly measuring the amount of the reaction product itself but also measuring the amount of a substance having a relationship with the amount of the reaction product. The amount of analyte to be determined in the sample can be calculated from the amount of reaction product determined as described above. In addition, qualitative assays that determine the presence or absence of a reaction product are also within the scope of the present invention for determining a reaction product.
In addition, when measurements are performed using whole blood, hematocrit correction is typically required after the measurement. In most samples, the hematocrit becomes about 40 to 50%. In addition, when a qualitative determination is made as an item of determination of positive or negative determination in the case of an infectious disease, hematocrit is less important. Therefore, there is no practical problem even when the hematocrit value is not measured for each sample. When the hematocrit value is available, a more accurate test result may of course be obtained by performing hematocrit correction (test result X100/(100-hematocrit value (%))).
The kit of the present invention is a kit for measuring an analyte in whole blood, which comprises a first substance carried by a solid carrier and specifically binding to the analyte, a second substance specifically binding to the analyte, and a detergent which does not cause hemolysis and does not inhibit the reaction of the analyte with the first and second substances when mixed with whole blood. The kit of the present invention has the same composition as a conventional kit for measuring an analyte in plasma or serum, except that it includes the aforementioned detergent. That is, the kit of the present invention is used in the aforementioned test method of the present invention.
The kit preferably further comprises a whole blood treatment solution. The whole blood treatment solution may contain a detergent as described above. As an arbitrary component, the kit may further include a reaction diluent, a substrate solution, a substrate dissolving solution, a washing solution, a reaction terminating solution, and the like. By using the kit, the test method of the invention can be rapidly and conveniently carried out, and has good accuracy and stability.
The test method of the present invention can be performed using an instrument, a cartridge, or the like used in known automatic tests. Specific examples thereof include cartridges and instruments described in WO01/84152, Japanese patent Laid-open No. 11-316226 and the like. In addition, the kit of the present invention is also packaged in a cartridge used in an automatic test, and is suitably used in the aforementioned automatic test instrument. By combining the kit of the present invention with an instrument and cartridge for automatic testing, a rapid, convenient and highly sensitive test method can be provided.
Best mode for carrying out the invention
The present invention will be explained in more detail with reference to the following examples. However, the scope of the present invention is not limited by these examples.
Example 1: preparation of a chemiluminescent enzyme immunoassay reagent for hepatitis B Virus surface antigen (HBsAg)
(1) Preparation of magnetic particles
The anti-HBsAg polyclonal antibody was physically attached to the magnetic particles (0.3 μ M) in 50mM phosphate buffer (pH4.0), and then treated with Tris buffer (0.1M, pH8.0) containing 0.2% BSA at 37 ℃ for 1 day to give anti-HBsAg antibody-binding particles. The magnetic particles obtained were suspended in 0.1M Tris buffer (pH8.0) at a concentration of 100 to 200. mu.g/ml.
(2) Preparation of labeled antibodies
The anti-HBsAg monoclonal antibody was conjugated to calf alkaline phosphatase (ALP) by the maleimide method to give an ALP-labeled anti-HBsAg antibody. The obtained labeled antibody was suspended in 0.1M Tris buffer (pH8.0) at a concentration of 0.2 to 0.5. mu.g/ml.
(3) Preparation of B/F Wash solution
A0.1 Mtris buffer (pH8.0) containing 1% Tween 20 and 0.15M NaCl was prepared.
(4) Luminescent substrate
As a luminescent substrate, 25mM AMPPD solution (Tropix) was used.
Example 2: anti-HBsAg antibody binding particles and assay for labeled antibodies
First, the potency of the reagent produced in example 1 was confirmed. The potency was evaluated using HBsAg positive control serum and negative control serum as samples, and the potency was evaluated without using whole blood. In the test, 60. mu.l of sample was added with 150. mu.l of magnetic particles, stirred and incubated at 42 ℃ for 10 minutes. Then, the magnetic particles were collected with a magnet and washed with a B/F washing solution. Subsequently, the washed magnetic particles were added with 150. mu.l of labeled antibody, stirred, incubated at 42 ℃ for another 10 minutes, and then the magnetic particles were collected with a magnet and washed well with a B/F washing solution. In addition, the washed magnetic particles were added with 200. mu.l of AMPPD solution, mixed well and incubated at 42 ℃ for 5 minutes. Then, the emission intensity was measured by a photomultiplier tube (PMT).
The above test was repeated for 12 days, and the reproducibility in the measurement per day was examined. As a result, favorable results were obtained as shown in table 1.
TABLE 1
Example 3: detecting Whole blood treatment solution
The detergent is set to be added to the reaction system by previously adding the detergent to the whole blood treatment solution, and thus the measurement is performed. Detergents suitable for the detection method of the present invention were detected by preparing whole blood treatment solutions by dissolving various detergents in 0.1M Tris buffer (pH8.0) containing 1% BSA and 0.15M NaCl.
Blood was collected using EDTA blood collection tubes. Then, 1U/ml of HBsAg was added to each whole blood left at 4 ℃ for 3 days and each plasma obtained by centrifuging the whole blood, and an HBsAg recovery experiment was performed using the emission intensity obtained in the test using plasma as 100%. The blood cell fraction of the whole blood, which precipitated during standing at 4 ℃, was slightly hemolyzed in the plasma fraction. The amount of hemolyzed cells was measured by another method, and it was found that about 5% of the total red blood cells were hemolyzed.
The test was carried out in the same manner as in example 2. The mixing ratio of the whole blood and each of the whole blood treatment solutions was 9: 1, and the mixing ratio of the plasma and the purified water was 9: 1, and HBsAg was immediately measured. In addition, instead of the whole blood treatment solution, the whole blood was mixed with the aforementioned buffer (0.1M Tris buffer (pH8.0) containing 1% BSA and 0.15M NaCl) to which no detergent was added, and HBsAg was measured in the same manner. Nonspecific adhesion of magnetic particles to the reaction vessel (made of polypropylene) and nonspecific aggregation of magnetic particles during the reaction can be confirmed visually in the presence or absence of hemolysis in the reaction system. The results are shown in Table 2.
TABLE 2
| Sample (I) | Concentration of detergent after mixing of whole blood | Hemolysis of blood | Adhesion of magnetic particles to the walls of the reaction vessel | Aggregation of magnetic particles | Luminous intensity | Recovery ratio | |
| Triton X-100 | Whole blood | 1％ | Exist of | Is not provided with | Exist of | 5130 | 42％ |
| Tween 20 | Whole blood | 1％ | Is not provided with | Is not provided with | Is not provided with | 10620 | 87％ |
| Tween80 | Whole blood | 1％ | Is not provided with | Is not provided with | Is not provided with | 10260 | 85％ |
| 3- (1-pyrido) -1-propanesulfonic acid salt | Whole blood | 2％ | Is not provided with | Trace amount of | Is not provided with | 10830 | 88％ |
| Brij78 | Whole blood | 1％ | Exist of | Is not provided with | Exist of | 5820 | 48％ |
| Saponin | Whole blood | 1％ | Exist of | Is obvious | Exist of | 9460 | 77％ |
| SDS | Whole blood | 1％ | Exist of | Is not provided with | Exist of | 2450 | 20％ |
| CHAPS | Whole blood | 1％ | Exist of | Is not provided with | Exist of | 8020 | 66％ |
| No detergent | Whole blood | 0％ | Is not provided with | Is obvious | Is obvious | 11690 | 96％ |
| Distilled water | Blood plasma | 0％ | Is not provided with | Is not provided with | Is not provided with | 12240 | 100％ |
Triton X-100: polyoxyethylene octyl phenyl ester
Tween 20: polyoxyethylene sorbitan mono-cinnamate
Tween 80: polyoxyethylene sorbitan monooleate
Brj 78: polyoxyethylene stearate
SDS (sodium dodecyl sulfate): sodium dodecyl sulfate
CHAPS: 3- { (3-Cholamidopropyl) dimethylamino } -1-propanesulfonic acid salt
As shown in Table 2, when whole blood was used as a sample, the recovery ratio was measured to be 85% or more in a sample mixed with Tween 20, Tween80 or 3- (1-pyrido) -1-propanesulfonate, or in a sample containing no detergent (0.1M Tris buffer (pH8.0) containing 1% BSA and 0.15M NaCl). Among these, the recovery ratio was the best in the sample containing no detergent. However, a very large amount of magnetic particles adhered to the inner wall of the reaction vessel, so that the B/F washing was not well performed, and therefore, it was not considered that an accurate test result had been obtained. Therefore, the samples mixed with Tween 20, Tween80 and 3- (1-pyrido) -1-propanesulfonate were then further examined.
In addition, it was confirmed in this experiment that, by the technique as described above, it is possible to easily select from various detergents, the concentration and type of which do not substantially cause hemolysis, do not inhibit an analyte and a substance that specifically binds to the analyte, and can prevent the influence of a component present in a reaction system of interest on the reaction system.
Example 4: detection of detergent type and concentration Using Whole blood
Blood was collected using a blood collection tube treated with heparin as an anticoagulant. To whole blood stored overnight at 4 ℃ and plasma obtained from the whole blood, 0.5U/ml of HBsAg was added, and the addition and recovery test method was performed in the same manner as in example 3 using 100% luminous intensity of plasma. For the whole blood treatment solution, comparisons were made using tween 20, tween80 and 3- (1-pyrido) -1-propanesulfonate selected from example 3, and triton X-100. Each detergent was added at final concentrations of 0.01, 0.1, 0.5, 1 and 10% after mixing with whole blood. Non-specific adhesion of magnetic particles to a reaction vessel (made of polypropylene) and non-specific aggregation of magnetic particles during the reaction were confirmed by visually observing each detergent in the reaction system in the presence or absence of hemolysis in the reaction system, and the recovery ratio with respect to the charged amount was determined. The results are shown in Table 3.
According to the results of the test, a good recovery ratio was selected for the amount added, without causing hemolysis or non-specific adhesion of the magnetic particles to the reaction vessel. As a result, particularly good results were obtained when Tween80 was added at a concentration of 0.5 to 10% or 3- (1-pyrido) -1-propanesulfonic acid salt was added at a concentration of 1%. When triton X-100 was added at a concentration of 0.5%, hemolysis was observed although a recovery ratio of 75% was generally advantageous. In addition, when tween 20 was added at a concentration of 1 to 10%, although hemolysis and non-specific adhesion of magnetic particles on the reaction vessel were not observed, a sufficient recovery ratio with respect to the amount added could not be obtained.
TABLE 3
| Sample (I) | Detergent concentration after mixing of Whole blood (%) | Hemolysis of blood | Adhesion of magnetic particles to the walls of the reaction vessel | Aggregation of magnetic particles | Luminous intensity | Recovery ratio | |
| Musical compositionGeneral formula X-100 | Whole blood | 0.01 | Is not provided with | Is provided with | Is provided with | 1084 | 19％ |
| Qutong-100 | Whole blood | 0.1 | Is not provided with | Is provided with | Is provided with | 1424 | 25％ |
| Qutong-100 | Whole blood | 0.5 | Is provided with | Is not provided with | Is provided with | 4288 | 75％ |
| Qutong-100 | Whole blood | 1 | Is provided with | Is not provided with | Is provided with | 2800 | 49％ |
| Qutong-100 | Whole blood | 10 | Is provided with | Is not provided with | Is provided with | 1422 | 25％ |
| Tween 20 | Whole blood | 0.01 | Is not provided with | Is provided with | Is provided with | 992 | 17％ |
| Tween 20 | Whole blood | 0.1 | Is not provided with | Is provided with | Is provided with | 1268 | 22％ |
| Tween 20 | Whole blood | 0.5 | Is not provided with | Is provided with | Is provided with | 2696 | 47％ |
| Tween 20 | Whole blood | 1 | Is not provided with | Is not provided with | Is not provided with | 3520 | 62％ |
| Tween 20 | Whole blood | 10 | Is not provided with | Is not provided with | Is not provided with | 2368 | 42％ |
| Tween80 | Whole blood | 0.01 | Is not provided with | Is provided with | Is provided with | 1304 | 23％ |
| Tween80 | Whole blood | 0.1 | Is not provided with | Is provided with | Is provided with | 1436 | 25％ |
| Tween80 | Whole blood | 0.5 | Is not provided with | Is not provided with | Is provided with | 4256 | 75％ |
| Tween80 | Whole blood | 1 | Is not provided with | Is not provided with | Is not provided with | 4564 | 80％ |
| Tween80 | Whole blood | 10 | Is not provided with | Is not provided with | Is not provided with | 5180 | 91％ |
| 3- (1-pyrido) -1-propanesulfonic acid salt | Whole blood | 0.01 | Is not provided with | Is provided with | Is provided with | 1596 | 28％ |
| 3- (1-pyrido) -1-propanesulfonic acid salt | Whole blood | 0.1 | Is not provided with | Is provided with | Is provided with | 2920 | 51％ |
| 3- (1-pyrido) -1-propanesulfonic acid salt | Whole blood | 0.5 | Is not provided with | Is not provided with | Is provided with | 2720 | 48％ |
| 3- (1-pyrido) -1-propanesulfonic acid salt | Whole blood | 1 | Is not provided with | Is not provided with | Is not provided with | 4896 | 86％ |
| 3- (1-pyrido) -1-propanesulfonic acid salt | Whole blood | 10 | Is provided with | Is not provided with | Is not provided with | 1272 | 22％ |
| Distilled water | Whole blood | 0 | Is not provided with | Is provided with | Is provided with | 788 | 14％ |
| Distilled water | Blood plasma | 0 | Is not provided with | Is not provided with | Is not provided with | 5696 | 100％ |
Example 5: assay with fresh Whole blood
Subsequently, a 3- (1-pyrido) -1-propanesulfonate selected from example 4 and Tween80 were assayed using fresh blood. In an emergency test, in particular, a test is required immediately after the blood is collected, and red blood cells in the whole blood may gradually become hemolyzed during storage, possibly affecting the test. Therefore, immediately after blood collection, testing was performed with fresh blood. The experiment for addition and recovery of HBsAg was performed in the same manner as in example 3. The whole blood treatment solution was prepared by adding 3- (1-pyrido) -1-propanesulfonate, tween80 and a mixture thereof. The results are shown in Table 4.
TABLE 4
| Sample (I) | Detergent concentration after mixing of Whole blood (%) | Hemolysis of blood | Adhesion of magnetic particles to the walls of the reaction vessel | Aggregation of magnetic particles | Luminous intensity | Recovery ratio | |
| 3- (1-pyrido) -1-propanesulfonic acid salt | Whole blood | 2％ | Is not provided with | Is not provided with | Is provided with | 11370 | 86％ |
| Tween80 | Whole blood | 1％ | Is not provided with | Is not provided with | Is not provided with | 13460 | 102％ |
| 3- (1-pyrido) -1-propanesulfonate + Tween80 | Whole blood | 2％1％ | Is not provided with | Is not provided with | Is not provided with | 12910 | 98％ |
| Distilled water | Blood plasma | 0％ | Is not provided with | Is not provided with | Is not provided with | 13180 | 100％ |
As a result of the test, a good ratio of 86 to 102% was obtained even when fresh whole blood was used.
Industrial applicability
According to the method of the present invention, an analyte can be tested at high sensitivity quickly and conveniently using whole blood as a sample.
Claims (9)
1. A method for testing an analyte in whole blood, which comprises a dilution step of diluting whole blood by mixing whole blood with a whole blood treatment solution, a reaction step of forming a reaction system including the diluted whole blood, a first substance carried by a magnetic particle and specifically binding to an analyte contained in the diluted whole blood, and a second substance labeled with an enzyme or a luminescent substance and specifically binding to the analyte, and reacting the analyte with the first and second substances to form a reaction product, a separation step of separating the reaction product formed, and a measurement step of testing the separated reaction product, wherein:
(1) the reaction step is carried out in a state where the blood cells are not destroyed; and
(2) the whole blood treatment solution contains a sufficient amount of a detergent, wherein the detergent is selected from the group consisting of polyoxyethylene sorbitan ester-type detergents and sulfobetaine-type detergents.
2. The method of claim 1, wherein the detergent is selected from the group consisting of Tween 20, Tween80 and 3- (1-pyrido) -1-propanesulfonate.
3. The method as claimed in claim 1, wherein the concentration of the detergent in the reaction system is in the range of 0.1 to 10%.
4. The method of claim 1, wherein the ratio of whole blood to whole blood processing solution is in the range of 99: 1 to 5: 95.
5. The method of claim 1, wherein the reacting step of reacting the analyte with the first and second substances comprises a first reacting step of reacting the first substance with diluted whole blood to form a first reaction product, and a second reacting step of reacting the second substance with the first reaction product to form a second reaction product.
6. The method of claim 1, wherein the first and second substances that specifically bind to the analyte are antigens or antibodies.
7. A method for determining an analyte in whole blood, comprising:
(1) a dilution step of mixing whole blood with the whole blood treatment solution to dilute the whole blood;
(2) a first reaction step of adding a first substance carried by magnetic particles and specifically binding to the analyte to diluted whole blood to react them to form a first reaction product in a reaction system;
(3) a first separation step of separating a first reaction product formed in the first reaction step from the reaction system;
(4) a second reaction step of adding a second substance labeled with an enzyme or a luminescent substance and specifically binding to the analyte to the separated first reaction product and reacting them to form a second reaction product in the reaction system;
(5) a second separation step of separating a second reaction product formed in the second reaction step from the reaction system; and
(6) a measuring step of measuring the separated second reaction product,
wherein the whole blood treatment solution contains a sufficient amount of a detergent selected from the group consisting of polyoxyethylene sorbitan ester-type detergents and sulfobetaine-type detergents.
8. The method of claim 7, wherein the detergent is selected from the group consisting of Tween 20, Tween80 and 3- (1-pyrido) -1-propanesulfonate.
9. The method of claim 7, wherein the first and second substances that specifically bind to the analyte are antigens or antibodies.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2001067360 | 2001-03-09 | ||
| JP67360/2001 | 2001-03-09 | ||
| PCT/JP2002/002139 WO2002073203A1 (en) | 2001-03-09 | 2002-03-07 | Method of measuring whole blood |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| HK1066861A1 HK1066861A1 (en) | 2005-04-01 |
| HK1066861B true HK1066861B (en) | 2009-06-05 |
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